The purpose of this work is to elucidate the biochemical and molecular mechanisms responsible for prolactin (PRL) and growth hormone (GH) secretion. In particular, these efforts will be directed toward assessing the relationship between thiols and disulfides and the integrity of adenohypophysial secretory granules that store the hormones as disulfide bonded oligomers. I have carried out several preliminary studies: a granule membrane anion-sensitive Mg+2-ATPase has been characterized; ATP and anions have been shown to stimulate hormone release from isolated granules, and divalent metals to inhibit; a cytosolic glutathione (GSH):disulfide oxidoreductase (thioltransferase) has been partially purified and shown to utilize GH, PRL, and granule membrane disulfides as substrates; glutathione reductase and GSH S-transferase activities have been identified; thiol-stimulated neutral protease activity has been found in granule membranes; and my most recent experiments have revealed that low concentrations of added thiols markedly stimulate hormone release from isolated granules, whereas disulfides and thiol-blocking agents diminish release. The proposed studies will examine thiol:disulfide interchange to ascertain if this contributes to the stability of granule structure. Membrane protein thiols available to thiol-blocking agents will be identified, and their importance with respect to release will be examined. Methods to test thiol and metal regulation of ATPase and the thiol-activated protease will allow correlation, if any, with changes in hormone release. In addition, I will measure cytosolic thiol concentrations and activities of enzymes involved in the control of GSH and other cofactors relevant to the pituitary redox state. I will assess the effects of thioltransferase, glutathione reductase, and GSH S-transferases on GSH levels, membrane thiol:disulfide equilibria, hormone oligomeric state, and release. Also, experiments will be devised to define thiol influences on binding of granules to cellular components including microtubules and plasma membranes, and to dopamine, TRH, and somatostatin. Methods will employ radioimmunoassay, inhibitor studies, and isolation and purification techniques based on charge, size, and site-specificity. Thus, it is hoped that a biochemical approach refining our knowledge of an isolated granule system will shed light on the molecular mechanisms of GH and PRL secretion.